Journal: Molecular Cancer Research

Article Title: Progesterone and 1,25-Dihydroxyvitamin D3 Inhibit Endometrial Cancer Cell Growth by Upregulating Semaphorin 3B and Semaphorin 3F

doi: 10.1158/1541-7786.mcr-11-0213

Figure Lengend Snippet: Figure 1. Expression of SEMA3B, SEMA3F, plexin A1, plexin A3, NP1, and NP2 in human endometrial tumors. Immunohistochemical analysis of endometrial tumors from TMA using SEMA3B, SEMA3F, plexin A1, plexin A3, NP1, and NP2 antibodies was done. Expression of SEMA3B (2) and SEMA3F (7) was significantly high in normal endometrial tissues. Gradual decrease of SEMA3B (3–4) and SEMA3F (8–9) expression was seen in grades I–II. Grade III (5 and 10) showed no to weak expression of SEMA3B and SEMA3F. Negative controls for SEMA3B (1) and SEMA3F (6) are shown. Expression of plexin A1 (12) and plexin A3 (17) was significantly high in normal endometrial tissues. No change in plexin A1 expression was noticed in different grades of endometrial cancer. Gradual decrease of plexin A3 expression was seen in grades I to III (18–20). Negative controls for plexin A1 (11) and plexin A3 (16) are shown. No alterations of NP1 (22) and NP2 (27) in normal and different grades of endometrial cancer tissues were seen. Original magnification, 400.

Article Snippet: To better understand the role of SEMA3B and SEMA3F in the inhibition of proliferation, cells were transfected with siRNAs directed against NP1 and NP2 (5 nmol/L; Santa Cruz Biotechnology, Inc.) or scrambled siRNA and treated 24 hours later with SEMA3F-CM or SEMA3B-CM for 72 hours.

Techniques: Expressing, Immunohistochemical staining

Journal: Molecular Cancer Research

Article Title: Progesterone and 1,25-Dihydroxyvitamin D3 Inhibit Endometrial Cancer Cell Growth by Upregulating Semaphorin 3B and Semaphorin 3F

doi: 10.1158/1541-7786.mcr-11-0213

Figure Lengend Snippet: Figure 2. Upregulation of SEMA3B and SEMA3F in endometrial cancer cells by P4 and 1,25(OH)2D3. A, normal immortalized EM-E6/E7/TERT and endometrial cancer cells (Ishikawa, HEC-1B, and RL-95) were evaluated by Western blot for basal expression of SEMA3B, SEMA3F, plexin A1, plexin A3, NP1, and NP2. Cells and CM were harvested, and 20 mg of protein from the whole-cell extract or CM were loaded in each lane. The blot was probed with the indicated antibody. b-Actin was used as a loading control. B, mouse E16 cerebellum and rat brain lysates were used as positive controls for SEMA3B and SEMA3F, respectively. Lysates probed with only secondary antibody (rabbit IgG) were used as negative controls. C, effect of P4 and 1,25(OH)2D3 on endometrial cancer cell (Ishikawa, HEC-1B, and RL-95) growth and apoptosis. The cells were treated with P4 (12.5–200 mmol/L) or 1,25(OH)2D3 (50–400 nmol/L) for 72 hours, and the viability of cells was determined by MTT assay. Data shown are mean SD of 3 separate experiments in which each treatment was repeated in 8 wells. The cells were treated with vehicle only or specified concentrations of P4 and 1,25(OH)2D3 for 72 hours, harvested, and cell lysates were prepared for caspase-3 enzyme activity. The data are representative of 3 independent experiments with similar results. , statistically significant changes in cell viability/apoptosis, compared with those seen in control cells (P < 0.05). D, the effect of P4 (25 mmol/L) and 1,25(OH)2D3 (100 nmol/L) on the expression of SEMA3B and SEMA3F was studied in endometrial cancer cell lines. Cells were cultured with P4 or 1,25(OH)2D3 in the presence or absence of antagonists (mifepristone for P4 and telmisartan for 1,25(OH)2D3) for 72 hours. Protein was extracted from cell cultures and loaded in each lane and blots were probed with SEMA3B and SEMA3F. b-Actin was used as a loading control.

Article Snippet: To better understand the role of SEMA3B and SEMA3F in the inhibition of proliferation, cells were transfected with siRNAs directed against NP1 and NP2 (5 nmol/L; Santa Cruz Biotechnology, Inc.) or scrambled siRNA and treated 24 hours later with SEMA3F-CM or SEMA3B-CM for 72 hours.

Techniques: Western Blot, Expressing, Control, MTT Assay, Activity Assay, Cell Culture

Journal: Molecular Cancer Research

Article Title: Progesterone and 1,25-Dihydroxyvitamin D3 Inhibit Endometrial Cancer Cell Growth by Upregulating Semaphorin 3B and Semaphorin 3F

doi: 10.1158/1541-7786.mcr-11-0213

Figure Lengend Snippet: Figure 5. Expression of NP2 is required for SEMA3F and SEMA3B inhibition of MMPs. Endometrial cancer cells (HEC-1B and Ishikawa) were transfected with a scrambled siRNA, siRNA for NP1, siRNA for NP2 or both. Transfected cells were exposed to SEMA3F-CM, or SEMA3B-CM or control-CM for 72 hours. A, cells extracts were analyzed by Western blotting for the expression of MMP-9 and MMP-2. b-Actin was used as a loading control. B, cell viability was assessed by MTS assay. Columns, mean; bars, SD. , P < 0.05, significant comparisons between the different treatments shown. C, the gelatinolytic activity of MMP-2 and MMP-9 in CM of endometrial cancer cells. Endometrial cancer cells overexpressing SEMA3B or SEMA3F or mock empty vector–transfected cells were serum starved for 12 hours and then incubated in the serum-free culture medium for 24 hours. CM were collected and then subjected to gelatin zymography. Representative picture from 3 independent experiments is shown. D, Western blot for MMP-9 and MMP-2 in HEC-1B and Ishikawa cells after NP1 siRNA, NP2 siRNA, both NP1 and NP2 siRNAs, or control siRNA knockdown followed by treatment with SEMA3F-CM or SEMA3B-CM for 72 hours. E, total cellular protein extracts were analyzed for aVb3 integrin using specific antibodies. b-Actin was used as a loading control.

Article Snippet: To better understand the role of SEMA3B and SEMA3F in the inhibition of proliferation, cells were transfected with siRNAs directed against NP1 and NP2 (5 nmol/L; Santa Cruz Biotechnology, Inc.) or scrambled siRNA and treated 24 hours later with SEMA3F-CM or SEMA3B-CM for 72 hours.

Techniques: Expressing, Inhibition, Transfection, Control, Western Blot, MTS Assay, Activity Assay, Plasmid Preparation, Incubation, Zymography, Knockdown